Recent progress in molecular biology has led to the development of gene therapy as a new treatment strategy for cardiovascular diseases. Targeted diseases range from single gene deficiency diseases to more complex diseases in adults, such as peripheral arterial diseases. For example, critical limb ischemia is estimated to develop in 500 to 1000 per million individuals in one year (“Second European Consensus Document on Chronic Critical Leg Ischemia.”, Circulation 84(4 Suppl.) IV 1-26 (1991)). In patients with critical limb ischemia, amputation, despite its associated morbidity, mortality and functional implications, is often recommended as a solution against disabling symptoms (M. R. Tyrrell et al., Br. J. Surg. 80: 177-180 (1993); M. Eneroth et al., Int. Orthop. 16: 383-387 (1992)). There exists no optimal medical therapy for critical limb ischemia (Circulation 84(4 Suppl.): IV 1-26 (1991)).
Recently, the efficacy of therapeutic angiogenesis by gene transfer of vascular endothelial growth factor (VEGF) has been reported to be effective for human patients with critical limb ischemia (I. Baumgartner et al., Circulation 97: 1114-1123 (1998); J. M. Isner et al., J. Vasc. Surg. 28: 964-973 (1998); I. Baumgartner et al., Ann. Intern. Med. 132: 880-884 (2000)) and myocardial ischemia (D. W. Losordo et al., Circulation 98: 2800-2804 (1998); P. R. Vale et al., Circulation 102: 965-974 (2000); T. K. Rosengart et al., Circulation 100: 468-474 (1999); T. K. Rosengart et al., Ann. Surg. 230: 466-470 (1999)). In addition to VEGF, gene transfer of other angiogenic growth factors, including fibroblast growth factor (FGF), hepatocyte growth factor (HGF) and hypoxia-inducible factor (HIF), has also been reported to stimulate collateral formation (Y. Taniyama et al., Gene Ther. 8: 181-189 (2000); H. Tabata et al., Cardiovasc. Res. 35: 470-479 (1997); H. Ueno et al., Arterioscler. Thromb. Vasc. Biol. 17: 2453-2460 (1997); K. A. Vincent et al., Circulation 102: 2255-2261 (2000); F. J. Giordano et al., Nat. Med. 2: 534-539 (1996); M. Aoki et al., Gene Ther. 7: 417-427 (2000); H. Ueda et al., Ann. Thorac. Surg. 67: 1726-1731 (1999); E. R. Schwarz et al., J. Am. Coll. Cardiol. 35: 1323-1330 (2000)).
The feasibility of gene therapy using angiogenic growth factors to treat peripheral arterial disease seems to be superior to recombinant protein therapy. For example, through gene therapy, one can potentially maintain an optimally high and local concentration over time. Thus, in the case of therapeutic angiogenesis, to avoid side effects, it may be desirable to deliver a lower dose of protein through an actively expressed transgene in the artery over a period of several days or more, rather than administering a single or multiple bolus doses of recombinant protein. Interestingly, most successful clinical trials treating peripheral arterial diseases using angiogenic growth factors have involved intramuscular transfection of naked plasmid DNA (I. Baumgartner et al., Circulation 97: 1114-1123 (1998); J. M. Isner et al., J. Vasc. Surg. 28: 964-973 (1998); I. Baumgartner et al., Ann. Intern. Med. 132: 880-884 (2000); D. W. Losordo et al., Circulation 98: 2800-2804 (1998); P. R. Vale et al., Circulation 102: 965-974 (2000)). However, such in vivo gene transfer, by direct injection of “naked” plasmid DNA into skeletal muscle, has been known to be inefficient.
Therefore, more efficient methods for gene transfer are required in the art for therapeutic application. Thus, many investigators have been focusing on alternate methods, such as the adenoviral gene transfer method (H. Ueno et al. Arterioscler. Thromb. Vasc. Biol. 17: 2453-2460 (1997); F. J. Giordano et al., Nat. Med. 2: 534-539 (1996); D. F. Lazarous et al., Cardiovasc. Res. 44: 294-302 (1999); L. Y. Lee et al., Ann. Thorac. Surg. 69: 14-23 (2000); L. H. Gowdak et al., Circulation 102: 565-571 (2000); O. Varenne et al., Hum. Gene Ther. 10:1105-1115 (1999); E. Barr et al., Gene Ther. 1: 51-58 (1994)). Although adenoviral vectors are efficient (H. Ueno et al., Arterioscler. Thromb. Vasc. Biol. 17: 2453-2460 (1997); F. J. Giordano et al., Nat. Med. 2: 534-539 (1996); D. F. Lazarous et al., Cardiovasc. Res. 44: 294-302 (1999); L. Y. Lee et al., Ann. Thorac. Surg. 69: 14-23 (2000); L. H. Gowdak et al., Circulation 102: 565-571 (2000); O. Varenne et al., Hum. Gene Ther. 10:1105-1115 (1999); E. Barr et al., Gene Ther. 1: 51-58 (1994)), they have some theoretical disadvantages, such as induction of strong immunogenicity in the host (V. J. Dzau et al., Proc. Natl. Acad. Sci. USA 93: 11421-11425 (1996)). In addition to efficiency, safety is also an important issue for gene transfer methods. The infusion of adenovirus has recently been reported to cause deleterious side effects (E. Marshall, Science 286: 2244-2245 (1999)). Thus, in the interests of safety, it would be more desirable to make non-virus-mediated plasmid DNA more efficient to achieve an ideal treatment for peripheral arterial diseases. Such innovation in plasmid DNA-based gene transfer should provide methods with high transfection efficiency without severe side effects.
To increase the transfection efficiency of naked plasmid DNA, the present inventors previously tested the use of ultrasound and echo contrast microbubbles (Optison® (FS069); Molecular Biosystems). As a result, the inventors discovered that high transfection efficiency could be achieved by ultrasound-mediated plasmid DNA transfection using echo contrast microbubbles (Y. Taniyama et al., Circulation 105: 1233-1239 (2002); Y. Taniyama et al., Gene Therapy 9: 372-380 (2002)). Using ultrasound exposure in the presence of microbubble echo contrast agents, approximately 300-fold increment in transgene expression following naked DNA transfection was reported in in vitro experiments (A. Lawrie et al., Gene Ther. 9: 372-380 (2002)) In addition, the inventors confirmed the usefulness of ultrasound-mediated plasmid DNA transfection with Optison® into rat skeletal muscle as well as rat carotid artery (Y. Taniyama et al., Circulation 105: 1233-1239 (2002); Y. Taniyama et al., Gene Therapy 9: 372-380 (2002)). Due to the appearance of transient holes in the cell membrane through the spreading of the bubbles, this method increased the transfection efficiency.